Immune responses contribute to the success of radiation therapy of solid tumours; however, the mechanism of triggering CD8+ T cell responses is poorly understood. Antigen cross-presentation from tumour cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8+ T cell stimulation. We established a cross-presentation model in prostate cancer in which DC present a naturally expressed oncofetal tumour antigen (5T4) from irradiated DU145 tumour cells to 5T4-specific T cells. Ionising radiation (12 Gy) caused G2/M cell cycle arrest and cell death, increased cellular 5T4 and high-mobility protein group-B1 (HMGB1) levels and upregulated surface calreticulin and Hsp70 expression in DU145 cells. Co-culture of DC with irradiated tumour cells lead to efficient phagocytosis of tumour cells and upregulation of CD86 and HLA-DR on DC. CD8+ 5T4-specific T cells, stimulated with these DC, proliferated and produced IFNγ. Inhibition of HMGB1 decreased T cell stimulation but not DC activation, while TRIF/MyD88 inhibition only had a marginal effect on T cell stimulation. Unlike previous reports, I found no functional evidence that DC with Asp299Gly toll-like receptor-4 (TLR4) single nucleotide polymorphism had impaired ability to cross-present tumour antigen. However, I observed a highly significant and robust prevention of antigen cross-presentation when tumour cells were pretreated with the novel Hsp70 inhibitor, VER 155008. The inhibitor also prevented CD86 upregulation on DC co-cultured with irradiated tumour cells. Together, the results in this thesis demonstrate that radiation induces immunologically relevant changes in tumour cells, which can trigger CD8+ T cell responses via a predominantly Hsp70-dependent antigen cross-presentation process.

Colorectal cancer (CRC) is one of the most common cancers in the Western world. Screening for CRC using faecal occult blood test (FOBT) is well established. There is evidence that DNA based stool tests may be more effective than FOBT. Hypothesis The hypothesis is that in patients with CRC, rectal mucus may contain DNA derived from colonic tumours. It is speculated that quantitative or qualitative assessment of DNA in rectal mucus may permit an improved method of CRC screening. Aims Using surgically resected specimens of colonic tumours: To assess the feasibility and reliability of measuring DNA in mucus samples To compare different devices to measure mucus DNA To assess the amount of mucus DNA at various distances from colonic tumours In patients with CRC and controls: To compare the amount of DNA in rectal mucus Using a panel of 3 DNA methylation markers, to compare the rectal mucus DNA methylation profile between patients with CRC and controls Methods Surgical colectomy specimens were obtained from 25 patients with CRC. The feasibility and repeatability of measuring mucus DNA amounts was established using different buffer solutions, different storage techniques and different sampling devices. Mucus DNA amounts were measured at tumour sites and various distances proximal and distal to the tumour. 58 patients referred to a colorectal outpatient clinic with suspected CRC were assessed. Rectal mucus samples were obtained using a balloon device introduced through a proctoscope. All patients were investigated by colonoscopy to clarify the presence or absence of a CRC. The amount of DNA in the mucus samples was measured. The presence of three DNA methylation markers (NDRG4, TFP12 and GATA4) was assessed in all samples. All studies were approved by the local ethics committee. Results Reliable measurement of DNA from mucus samples was established using balloon, foam and brush devices and a cell lysate buffer. Higher amounts of DNA in surgical specimens were found distal to tumours compared to proximally. In patients with CRC the amount of DNA in rectal mucus was higher than in controls (no disease or benign polyps). The three DNA methylation marker panel had a sensitivity of 87% and specificity of 27.5% for the detection of CRC. Conclusions The results are consistent with the hypothesis that DNA detected in rectal mucus is derived from proximal tumours. Higher levels of rectal mucus DNA are obtained from patients with CRC than from controls. The selected DNA methylation panel was not sufficiently useful in our sample group to be of use as a screening technique, due to poor specificity. Further work is in progress to compare DNA abnormalities in resected tumour tissue with DNA from rectal mucus in the same patients. Future work may be required to improve the panel of DNA abnormalities assessed.

Mutational activation of RAS is one of the most common molecular abnormalities associated with acute myeloid leukaemia (AML). Normal human haematopoietic progenitor cells (HPC) expressing mutant RAS overproduce ROS due to NADPH oxidase (NOX) activation and this promotes the proliferation of these cells as well as AML blasts. The mechanisms by which ROS promote proliferation is however unclear. The current study investigated the effect of RAS-induced ROS production on gene expression in normal HPC using gene expression profiling (GEP) and assessed whether ROS-induced gene expression changes contributed to the pro-proliferative phenotype. In order to determine the ROS-specific GEP, Affymetrix Human Exon 1.0ST arrays were used for the comparison of mutant RAS and control cells cultured in the presence or absence of the NOX inhibitor, DPI, which strongly suppressed the production of ROS. This analysis showed that RAS changed the expression of 342 genes. Of these, 24 genes were specifically altered in response to ROS production by these cells and amongst these increased expressions of genes of the glycolytic pathway were prominent. To establish the functional significance of up-regulated expression of glycolytic enzymes, aldolase C (ALDOC) was investigated since it showed greatest induction with ROS. ALDOC was directly induced by physiological levels of ROS in both HPC and AML cell lines. Further, overexpression of ALDOC demonstrated that its overexpression promoted the proliferation and serum-independent survival of leukaemic cell lines. Conversely, antiproliferative effects were observed when ALDOC was knocked-down in cells known to have high levels of constitutive ROS production. Given the high frequency of ROS production in AML, this study provides a plausible mechanism of enhanced glycolysis seen in this disease and suggests that agents restoring the redox environment could be used to correct metabolic imbalances which contribute to treatment resistance.

Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor ᴋB (RANK) and RANK ligand (RANKL), are members of the tumour necrosis factor receptor superfamily (TNFRSF), signal transducers which have pleiotropic actions. Each family member has unique structural attributes shown to couple them directly to specific signalling pathways involved in cell proliferation, differentiation and survival. Previous studies have clinically correlated OPG, RANK and RANKL expression, at both transcript and protein levels, with increasing cancer tumour burden, metastatic bone involvement and androgen status, however the mechanisms by which these molecules exert their effects remain elusive. This study aimed to establish what influence targeting OPG, RANK and RANKL expression may have on osteotrophic prostate and breast cancer cells in vitro and to subsequently explore the effect(s) Hepatocyte Growth Factor (HGF) and Bone Matrix Extract (BME) might also exert on cancer cell behaviour following manipulation of these molecules. The current study utilised 2 prostate cancer cell lines with varying androgen status, metastatic potential and bone metastasis phenotypes. Initial screening showed that the more aggressive osteolytic PC-3 cells expressed OPG, whilst weakly metastatic mixed-osseous LNCaP cells had very low expression. Whilst RANK was present in both cell lines, RANKL expression was only detected in the LNCaP cells. Reduction of OPG expression in the PC-3 cells resulted in increased cell invasion in vitro, which was further enhanced when treated with BME. No other cellular traits were affected by targeting OPG directly, however, cell migration was enhanced when the manipulated cells were exposed to the representative bone microenvironment. In contrast the addition of a recombinant form of OPG to LNCaP cells resulted in decreased cell invasion, a trend which was reversed when combined with BME. Combination of OPG and BME treatment reduced the migratory response of LNCaP cells, whilst combination of OPG and HGF were pro-migratory. The targeting of RANK in PC-3 cells affected cell proliferation and matrix adhesion in vitro though the addition of HGF or BME appeared to have no further direct influence on these manipulated cells. Targeting of the RANKL expression with a neutralising monoclonal antibody had little effect on cancer cell behaviour; however combined exposure with HGF or BME resulted in similar behaviour patterns seen under the OPG treatments. In our breast cancer cohort, RANK and RANKL expression were correlated with bone metastases and survival rates. Though OPG did not appear to be associated with grading, data also implied a role in overall survival. In the aggressive osteolytic MDA-MB-231 breast cancer cells, reduced OPG expression resulted in increased motility and invasion, traits which were little affected upon exposure to HGF or BME. In contrast the targeting of RANK expression in MDA-MB-231 cells resulted in reductions in all the cancer cell behaviours studied, but again these appeared unaffected under the influence of HGF or BME. The complexity of the bone environment underpins the vast number of soluble factors, signalling pathways and transcription regulators which can influence osteotrophic cancer cells. As indicated by the licensing of Denosumab, one therapeutic approach is not suitable for all osteotrophic cancers. Therefore further elucidation into the intricacies of these interactions is needed.

Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder that affects the development of haematopoietic cells and has an incidence of 2300 cases / year in the UK. Whilst treatment with conventional cytotoxic agents has significantly increased cure rates in AML, only 35-50% patients under the age of 60 years will be long term survivors. There is a need to develop novel agents targeting molecular lesions or dysregulated pathways to improve clinical responses. This study identified commonly dysregulated pathways and genes using Affymetrix gene expression profiling of AML patients. Analysis of gene expression data using MetacoreTM online gene ontology pathway analysis identified WNT proteins to be one of the most frequently dysregulated processes associated with AML. TCF7L2 was the most significantly dysregulated WNT transcription factor gene and was subsequently examined in greater detail. The expression of TCF7L2 was validated by RQPCR. These data significantly correlated with the normalised array expression data (R=0.748; P<0.01) which in turn correlated with overall protein expression determined and quantified by western blotting. TCF7L2 mRNA overexpression was found to be independently prognostic for reduced complete remission rate (P<0.05), OR=5.19 [95% C.I.=1.39 - 19.39]. The TCF7L2 gene undergoes exon splicing which yields multiple isoforms which have been reported to yield functionally distinct proteins. TCF7L2 mRNA isoform expression in AML patients was compared with that in normal human bone marrow and a human cord blood derived from CD34+ haematopoeitic progenitor cells. Extensive variability of TCF7L2 splicing at the 3’ end of the gene (exons 13-18) was identified. AML patients were heterogeneous in the isoform expression pattern, but aberrant exon composition (compared to normal cells) was not observed. At the protein level, expression of the 58 kDa isoform (exons 1-14 and 18) and 56 kDa isoform (exons 1-13 and 18) were detected. In both normal and AML cells, expression of the 56 kDa and 58 kDa isoforms were dominant. To determine the functional significance of TCF7L2 overexpression lentiviral shRNA vectors targeting TCF7L2 was transduced in leukaemic cell lines coexpressing a TCF-GFP reporter which enabled simultaneous analysis of the effect on TCFdependent transcription. Reporter activity was inhibited by shRNA vectors targeting TCF7L2, and the cells became non-responsive to WNT agonists (WNT3A and BIO) demonstrating that canonical WNT signalling is dependent on TCF7L2 expression in these cells. Phenotypically, shRNA expression was found to strongly inhibit proliferation and to promote apoptosis indicating that TCF7L2 expression is required for the proliferation and survival of myeloid leukaemia cells. Paradoxically, overexpression of individual TCF7L2 isoforms (72 kDa, 58 kDa and 56 kDa) suppressed WNT agonist responses in myeloid leukaemia cell lines but not in epithelial cells. Overexpression of TCF7L2 in normal CD34+ cells was found to promote monocytic differentiation. In summary, this study presents novel data identifying WNT signalling as the most commonly dysregulated process in AML. TCF7L2, a WNT transcription factor was found to be significantly overexpressed in AML and associated with poor clinical outcome. This protein was found to be required to maintain the proliferation and viability of myeloid leukaemia cells suggesting that targeting TCF7L2 maybe a valid approach in AML therapy.

Numerous factors influence an individual’s risk of colorectal cancer, including pathological features such as polyp size and multiplicity, and family history of colorectal malignancy. In clinical practice polyp size can be measured at different time points, however adenoma surveillance guidelines do not define which measurement to utilise, due to variance in data source. The initial study compared the measurements of 107 polyps. Variation in surveillance intervals occurred less frequently with postfixation than in situ measurements (5.6 versus 9.5%), supporting the use of post-fixation polyp size. A further study considered the level of agreement amongst histopathologists in Wales in the reporting of colorectal polyps. Only fair agreement (k = 0.24) was observed in the reporting of the completeness of excision. A lesion with epithelial misplacement and high grade dysplasia was misclassified as adenocarcinoma by five pathologists, indicating the need for further training and potential introduction of a formal accreditation process. Individuals with a moderate family history risk of colorectal cancer are at increased risk of colorectal lesions. Pathways through the Welsh genetics service were studied. 63.4% referrals were received from primary care. The majority of patient’s were female (70.8%). 93.8% patients were advised to undergo 5-yearly surveillance. Existing referral pathways were found to be complex increasing the risk of over/under surveillance. Little is known about colonoscopic surveillance outcomes following genetic assessment. A study of 172 patients revealed an adenoma detection rate (ADR) of 11.1% and advanced ADR of 4.1% at the index procedure. Cancer was diagnosed in 0.6% cases. The majority of lesions identified were diminutive low grade adenomas. Several endoscopic modalities have been utilised to enhance polyp detection in patients with a propensity to colonic polyps. Narrow band imaging was studied in 37 high-moderate risk patients, but did not significantly increase polyp yield above high definition white light colonoscopy.

This thesis examines the evidence base on which the NICE guideline for colorectal cancer was developed. The information supporting guidelines varies. Six separate studies researching the availability and quality of different types of such information were carried out. Methodology, epidemiology, clinical practice, diagnostic accuracy, therapeutic, and internationally sourced data was examined. The information was sourced by online data mining, national database queries, systematic reviewing of literature databases, and the observation of the NICE guideline development process through both membership of the guideline development group established to produce the recommendations, and the technical development team supporting its production. Results show that : • NICE methodology data is available publicly, is easily accessible online, and has been developed following an internationally accepted guideline quality assessment and development tool. • Epidemiology data on colorectal cancer is easily accessible and of good quality. • Data regarding current clinical practice on colorectal cancer collected by national databases has methodological challenges and is not easily accessible. • Diagnostic accuracy studies are less robustly developed comapared to therapeutic studies. Their design is heterogeneous making the results subject to bias and reporting is inconsistent. Quality assessment when evaluating diagnostic evidence for a guideline ensures clarity with regard to the strength of the recommendations. • Systematic reviews of therapeutic studies can be inappropriately considered high quality evidence using traditional quality appraisal and evidence classification methods. All outcomes of a study should be considered when assesing study quality and recommendations based on a more holistic grading of the evidence are a more accurate reflection of the evidence. • Evidence considered for guideline development is international in its nature and the national setting of a study does influence guideline recommendations. Overall, NICE methodology is of high quality. The research helps identify challenges that the evidence can present as a platform for future improvements.

Class n major histocompatibility complex antigens are cell surface ap-heterodimeric glycoproteins which function as important immune recognition molecules for the stimulation of CD4+-T lymphocyte-mediated immune responses. Their expression by tumour cells, and the possible implications, has been the subject of much debate. In this investigation aspects of the cytokine-mediated regulation of the transcription of the gene of the human class nMHC molecule lll..A-DRa were investigated in human cell lines derived from colorectal and neural tumours. The transcriptional regulation of HLA-DRa has been shown to be controlled through discrete promoter elements termed the W, X and Y boxes; the W and X elements were investigated here. Nuclear proteins were purified from inducible (colo 201, colo 205 and U373MG) and non-inducible (caco-2 and LS180) cell lines and were shown to associate with oligonucleotide probes corresponding to the W and X regulatory elements of the lll..A-DRa promoter using bandshift (gel retardation) experiments. Differences were observed in the populations ofW and X box-binding proteins between the cell lines. The IFN-y treatment of class n inducible colorectal cell lines resulted in the binding of different populations of transcription factors to these probes: novel factors were bound as well as proposed repressor proteins dissociating from these probes. When a promoter probe corresponding to the 470 base pairs upstream of the cap site was employed, differences in the binding of factors between inducible and non-inducible cell lines were again observed. Treatment of the colorectal tumour cell line colo 205 with IFN-y resulted in the appearance of a novel binding factor after 6hr treatment. Longer treatment resulted in the net loss of binding factors from the W-X box region in addition to the loss of a postulated "maintenance repressor factor" which associated less specifically with the W-X-Y box region. The use of reporter gene assays in the glioblastoma cell line U138MG showed that IFN-ap and TGF-p are both capable of suppression of IFN-y-induced CAT expression through a 680bp and a 320bp HLA-DRa promoter fragment. The levels of IFN-y-induced CAT expression were also greater for the 320bp promoter fragment expression vector. The suppression observed was greater with the larger promoter fragment. Concurrent IFN-y (IU/ml; 48hr) and IFN-ap (100U/ml; 48hr) reduced DRa680CAT expression to 47% and DRa320CAT expression to 84% of that observed with IFN-y alone; simultaneous treatment (with lU/ml IFN-y and 1000U/ml IFN-ap) of cells transiently transfected with pDRa320CAT resulted in CAT expression being 26% of that observed with IFN-y alone. For TGF-p (lOOU/ml) cotreatment with lU/ml IFN-y, the expression of CAT was 27% and 55% of that observed for IFN-y-treatment alone with pDRa680CAT and pDRa320CATtransfected U138MG, respectively. It was concluded that the 680bp fragment contained a repressor element absent form the truncated form. The 680bp fragment contained a putative IFN-ap response element which was removed by its truncation to yield the 320bp promoter region. The colorectal tumour cell lines were unable to express CAT driven by either HLA-DRa promoter fragment. It was hypothesised that a tissue-specific enhancer element was absent from both of these promoter fragments. This study demonstrated a variety of aspects of the cytokine-mediated regulation of HLA-DRa gene expression. Tissue-specific differences in the regulation of expression were shown between cells of neural and colorectal origin when they were interrogated with regards to their abilities for IFN-y-induced HLA-DRa promoter-driven gene expression. The binding of nuclear proteins to important regulatory elements of the HLA-DRa promoter also confirmed that tissue- and cell-line-specific differences occurred with this respect.

Cancer cells preferentially metabolise glucose via aerobic glycolysis (the Warburg effect), which is less energy efficient in teens of ATP production compared to oxidative phosphorylation (OXPHOS). Mitochondrial DNA (mtDNA) encodes proteins of the electron transfer chain and is crucial for functional OXPHOS. MtDNA exists as multiple copies in cells and, often in cancer, there is co-existence of mutant and wild-type mtDNA. There is evidence for mitochondria to contribute towards the tumor biology of multiple myeloma (MM) and glioblastoma multiforme (GBM). The mtDNA from both these cancer types were explored to determine its role in tumor biology. Sequencing of MM cells and tumor samples using the Ion Torrent next generation sequencer identified Cytochrome C Oxidase and ATP 6 to contain critical variants that are capable of disrupting protein function. Gene expression analysis determined that glycolysis is essential to maintaining MM cell proliferation. Without glycolysis, there was up-regulation in the expression of tumor survival genes, which was only effective in MM cells that had sufficient mtDNA copy numbers above the mtDNA set point. Sequencing of GBM cell lines, tumor and normal patient samples suggested that there is a predisposition of GBM tumors to acquire a set of GBM-specific mtDNA variants during tumor development. Conserved mtDNA regions, such as Cytochrome C Oxidase I, tend to be least susceptible to mutations. The presence of variants in these conserved regions carry more detrimental effects at the protein-level than at other mtDNA regions. Differentiation of GBM cells decreased the tumor phenotype, as assessed by gene expression analysis. Altogether, this thesis provides support for the importance of mtDNA in tumor biology. The implications are that the variants identified could be used to screen MM and GBM tumors in a clinical diagnostic lab for the treatment of both these cancer types.

This thesis is an exploration of the experiences and support needs of people who are affected by cancer. It begins with a systematic review of interventions that are designed to support children who have a parent with cancer. Critical consideration of the value of these interventions in providing desired outcomes for the child and their family is the focus of this paper. Ten evaluations, consisting of nine separate interventions were included for review. Support for the efficacy of these interventions in providing improvements across a variety of outcomes concerning child well-being, parental well-being and overall family functioning is indicated. However, these findings must be considered within the context of the methodological limitations of the current research body. Clinical implications with regard to United Kingdom service provision and intervention planning are discussed. The second paper reports on an Interpretative Phenomenological Analysis study exploring the experiences of people who have had a diagnosis of, and treatment for, malignant melanoma. Six participants were recruited to this study and interviewed using semi-structured interviews. Three overarching themes emerged from the data detailing the lived experiences for these participants. A tension between whether having melanoma was a serious life event or not, finding balance and dealing with the experience and reflections on the ongoing impact upon self typified these participants’ experiences. These findings are discussed with regard to the implications for clinicians working with people who have been diagnosed with, and treated for, melanoma. The final paper is a reflective account of the researcher’s research journey. Specific focus is afforded to the development of the researcher’s relationship with the topic area and the integral role of the participant interview process within this. The influence of the research experience in shaping the researcher’s identity as a clinician is also considered, as are indications for ongoing development.